Nmr spectrometer at 400 mhz

Informed consent was obtained from all patients and fasting blood samples were collected using EDTA tubes at the time of hospital visit. These were then immediately processed and frozen at − 80 °C until analysis. Plasma TMAO levels were determined using an NMR spectrometer at 400 MHz (Bruker, USA). The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was employed to obtain spectra (recycle delay-90°-t1-90°-tm-90°-acquisition) over 64 scans with four dummy scans. Quantification was achieved using a concentration of a known reference signal (in this case TSP) to determine the TMAO concentration. hs-cTnT was measured using a high-sensitivity (5^th generation) assay on a Roche Cobas e411 platform (Roche Diagnostics, Basel, Switzerland). We defined SMD as hs-cTnT ≥ 14 ng/L, as this is considered the upper limit (the 99^th percentile) of the normal range in the healthy population^{19 (link)}. eGFR was calculated using the modification of diet in renal disease (MDRD) equation.

Prior to the metabolomics analysis, the frozen serum samples were defrosted at 4°C and mixed. Then, the samples were centrifuged and 300 μl of supernatant was gently mixed with 300 μl of serum buffer (0.075 M Na₂HPO₄ pH 7.4 in D₂O_, 4.6 mM TSP, 0.004% NaN₃). This was followed by centrifugation at 10,000 g, 4°C for 10 min. The mixed samples, 550 μl, were transferred into 5 mm NMR tubes (DWK Life Sciences, Germany). These were kept at 4°C until analysis.
¹H-NMR spectra were acquired at 298 K using an NMR spectrometer at 400 MHz (Bruker, USA). The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was employed to obtain spectra (recycle delay-90°-t1-90°-tm-90°-acquisition) in 64 scans.